Project Summary/Abstract Protein sorting is the process in which newly made proteins are specifically delivered to their sites of action, which often involves crossing a membrane. Our research focuses on a unique and essential protein transport pathway found in the plasma membrane of bacteria and some archaebacteria and the chloroplasts and mitochondria of plants, the Twin Arginine Transport (TAT) system. TAT systems are of fundamental interest because they function in an entirely different manner from transport systems found in animal cells. Specifically, TAT systems transport fully folded and assembled proteins across ion tight membranes using only three membrane components, TatA, TatB, and TatC, and the protonmotive force. Despite the importance of the TAT system in bacteria and plants, the mechanism of transport of folded precursor is not well-characterized. Twin arginine transport (TAT) systems are of practical interest because many human pathogens export virulence factors, responsible for causing disease, via the Tat system. Tat systems are absent in animals while pathogens such as Mycobacterium tuberculosis (tuberculosis) and Helicobacter pylori (ulcers) rely on it for virulence. Thylakoids of plant chloroplasts, however, provide the most robust, reliable assay to gain mechanistic detail about Tat systems. Understanding the mechanism of Tat systems is crucial for rational design of agents to disrupt it in pathogenic bacteria. Therefore, studies of the Tat system mechanism in thylakoids will be of immense practical and economic value in the development of highly specific therapies for infectious diseases. Understanding the structure of the proteins in the membrane is fundamentally important for understanding the organization of the TAT transport complex, regardless of the organism that it is in. Understanding the arrangement of cpTAT proteins in thylakoid is fundamentally important for understanding the mechanism of transport by the TAT system. Knowing which components are near the mature domain of the precursor is fundamentally important for determining the conduit through which the precursor passes, which is not known. We will investigate the spontaneous membrane insertion of That4 and cpTatB using liposomes and dye-release methods as well as site-directed spin labeling and continuous wave electron paramagnetic resonance (cw-EPR) spectroscopy and pulsed-EPR to measure distances. The successful completion of these studies is expected to have an important impact in understanding protein transport specifically as it relates to membrane biogenesis and assembly of protein complexes. This proposal is focused on the detail of the transport mechanism; however, such focus promotes excellent training opportunities for undergraduate researcher to learn hypothesis driven research at all levels from identifying the problem to determining the best strategy for approach to the problem.